The broad objective of this renewal application is to continue to investigate relationships between cardiac metabolism and function. The major areas to be studied include: 1). the metabolic regulation of cardiac ATP-sensitive K+ (K.ATP) channels and further assessment of their role in cellular K+ loss during hypoxia and ischemia. Cellular K+ loss during acute myocardial ischemia is a major arrhythmogenic factor. One goal of these studies is to resolve how K.ATP channels, which are suppressed by [ATP]i in the micromolar range in excised patches, are activated sufficiently to account for cellular K+ loss during myocardial hypoxia and ischemia when [ATP]i remains well in the millimolar range. We will continue to investigate how the sensitivity of K.ATP channels to [ATP]i is extrinsically and possibly intrinsically modified by various components of the ischemic environment. We will also evaluate interactions between sulfonylurea antagonists and agonists of K.ATP channels and various components of the ischemic environment in order to understand the mechanisms of their effects on cellular K+ loss during hypoxia and ischemia, and to evaluate their potential as cardioprotective agents. 2). the mechanisms underlying functional compartmentation of glycolytic and oxidative metabolism in heart. Compartmentation of cardiac metabolism if it exists has important implications for strategies to prevent irreversible cardiac injury. Studies arising from the previous application demonstrated that anaerobic glycolysis preferentially suppressed cardiac K.ATP channels due to the association of key glycolytic enzymes with K.ATP channels in cardiac sarcolemma. We will further explore the mechanisms of the preferential dependence of K,ATP channels on glycolysis, specifically testing the hypothesis that the major function of glycolytic enzymes associated with K.ATP channels is to lower the free [ADP]i. We will also further explore the hypothesis that contractile function is preferentially supported by oxidative metabolism in heart. Finally we will further investigate the mechanisms underlying our observation that exogenous glucose utilization is superior to glycogenolysis at preserving cardiac function during hypoxia and reoxygenation in intact rabbit ventricle. 3). mechanisms of transsarcolemmal lactate movement in heart. We will quantitate the extent to which electrogenic transsarcolemmal lactate and K+ movement are coupled in heart, in order to evaluate the hypothesis that lactate-coupled K+ efflux is an important cause of increased cellular K+ efflux during early myocardial ischemia and hypoxia. To accomplish these goals the experimental approach will utilize standard patch clamp techniques in isolated intact and permeabilized ventricular myocytes, fluorescent pH indicators in patch-clamped myocytes, and ion-selective electrode and radioisotopic techniques in isolated intact arterially perfused interventricular septa. These studies will evaluate at a fundamental level how various aspects of cardiac function are regulated by cardiac metabolism. Knowledge of these interactions is essential to understand the response of heart muscle to ischemia and other metabolically impaired states and to provide new insights relevant to the protection of ischemic injury, currently the leading cause of death in our society.